Project Summary Ablation of germ cells (GCs) extends lifespan in various species, providing paradigms for tradeoffs between reproduction and longevity, modulation of aging by signaling between tissues, and responses to metabolic perturbation. We recently identified a critical mechanism in this pathway in C. elegans: a response to fat overload that occurs when lipids that were produced for reproduction are not consumed. In the digestive system counterpart (intestine), this lipid accumulates and induces nuclear localization of the transcription factor SKN-1, the Nrf protein ortholog. SKN-1 activates lipid metabolism genes, reduces fat levels, and increases lifespan. SKN-1 and Nrf proteins are well known to respond to stress from reactive oxygen species (ROS), but GC ablation activates SKN-1 through a different pathway that is largely uncharacterized. This novel pathway involves fatty acid (FA)-dependent signals, specific lipid transport and metabolism activities, the gasotransmitter H2S, and 150 genes we have identified by RNA intereference (RNAi) screening. It is possible that this pathway may be triggered by particular FAs, or by bioactive lipid mediators (LMs) derived from them. In C. elegans we have collaboratively detected low-abundance LMs that promote or resolve inflammation in humans. GC ablation appears to alter their profiles, suggesting an ?inflammatory?-like response. Our findings provide a new genetically tractable platform for (1) uncovering how organisms respond to lipid overload, and (2) elucidating how specific lipid-based signals regulate gene expression and possibly aging. They also may represent a paradigm for understanding how mammalian Nrf proteins protect against fatty liver disease. This project will address a number of exciting questions. Aim 1 will elucidate lipid-dependent effects of GC ablation. Analysis of tissue-specific gene expression in the intestine and studies of particular SKN-1 isoforms will reveal FA-dependent effects that are mediated by SKN-1, and other regulators. Studies of FA profiling and ?-oxidation will reveal whether SKN-1 mediates effects that correlate with these gene expression changes. Our LM detection effort will identify candidate LMs that are modulated by GC ablation, and will enable study of these important regulators in this genetically tractable organism. Aim 2 will reveal how GC ablation and lipid signals activate SKN-1 and possibly other regulators. A combination of FA biosynthesis enzyme knockdown with administration of specific FAs and LMs should identify specific lipids that mediate these effects. Completion of our RNAi screening will suggest additional mechanisms through which GC ablation activates SKN-1. Finally, in the centerpiece of this project, epistasis and model-driven experiments will place H2S and lipid signals within this new pathway, and will elucidate the involvement of candidate regulatory mechanisms that our results have identified. Completion of these aims will provide new insights into lipid-based gene regulation, control of SKN-1/Nrf, and longevity assurance, thereby having major impacts in the fields of aging, stress responses, lipid metabolism, and lipid-based signaling.